The aggregation properties of zinc-free insulin have been studied using static and dynamic light scattering. The aggregation has been investigated as a function of three parameters, the concentration of sodium chloride (in the range 10-100 mM), the pH value (in the range pH 7.5-10.5), and the insulin concentration (1.8-13.4 mg/mL). The measured homodyne autocorrelation function was used to determine the apparent mean hydrodynamic diameter as well as the apparent weight-averaged molar mass of the insulin species in solution. A method of data analysis was employed, which allows the separation of light scattering contributions from the insulin oligomers and from irrelevant macromolecules and possible impurities present in the sample solutions. Also, a simple phenomenological equilibrium model describing the association of oligomers of insulin is presented. One aspect of this model is that it makes it possible to determine weight average molar masses corrected for virial effects on the Rayleigh ratio. This was necessary because virial effects cannot be isolated and corrected for by dilution since this would change the equilibrium distribution of oligomers. The basis of the model is a positive contribution to Gibbs free energy from charge repulsion depending on the protein charge and the number of monomers in the oligomers, and an assumed constant negative contribution to Gibbs free energy arising from either an entropic gain or hydrogen bonding upon association. The equilibrium model gives a good description of both the apparent weight average molar masses and the apparent hydrodynamic diameters, when the effect of the insulin concentration is taken into account by including virial effects arising from charge-charge repulsion (Donnan effect). The result shows that the association of insulin as a function of pH and ionic strength can be described by an effective charge equal to the charge derived from proton titration reduced by the number of sodium ions binding to insulin. At the lowest pH and highest salt concentration (pH 7.5, 100 mM NaCl, 12 mg/mL insulin), the weight average molar mass is close to that of the hexamer, and at the highest pH and lowest salt concentration (pH 10.5, 10 mM NaCl, 1.9 mg/mL), the weight average molar mass is close to that of the monomer. In all cases, however, a distribution of oligomers is present with a relative Gaussian width of about 30%.(ABSTRACT TRUNCATED AT 400 WORDS)

The aggregation processes leading to crystallization and precipitation of canavalin have been investigated by dynamic light scattering (DLS) in photon correlation spectroscopy (PCS) mode. The sizes of aggregates formed under various conditions of pH, salt concentration, and protein concentrations were deduced from the correlation functions generated by the fluctuating intensity of light scattered by the solutions of the protein. Results obtained indicate that the barrier to crystallization of canavalin is the formation of the trimer, a species that has been characterized by x-ray crystallographic studies (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). The dimensions of the trimer in solution are in good agreement with those obtained both from the crystal (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480) and from a low angle x-ray scattering study in solution (Plietz, P., P. Damaschun, J. J. Müller, and B. Schlener. 1983. FEBS [Fed. Eur. Biochem. Soc.] Lett. 162:43-46). Furthermore, under conditions known to lead to the formation of rhombohedral crystals of canavalin, a limiting size is reached at high concentrations of canavalin. The size measured corresponds to an aggregate of trimers making a unit rhombohedral cell consistent with x-ray crystallographic data (McPherson, A. 1980. J. Biol. Chem. 255:10472-10480). Presumably, such aggregates are the nuclei from which crystal growth proceeds. The present study was undertaken primarily to test the potential of DLS (PCS) as a tool for rapid, routine screening to determine the ultimate fate of protein solutions (i.e., crystallization or amorphous precipitation) at an early stage, therefore eliminating the need for long-term visual observation. Achieving this goal would constitute amajor advance in the practive of protein crystallization. Delays imposed by visual observation would be considerably reduced, and a more systematic approach could be adopted to select experimental conditions.Our findings with canavalin demonstrate that DLS(PCS) is, indeed, a selective and sensitive probe of precrystallization conditions. Other advantages of this technique include the facts that it is noninvasive, nondestructive,universal, and does not require calibration.

Substrate analogues of L-serine have been found that react with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase. Upon reaction with alpha 2 beta 2, the analogues glycine, L-histidine, L-alanine, and D-histidine form chemical intermediates derived from reaction with enzyme-bound pyridoxal 5'-phosphate with characteristic UV-visible spectral bands. The spectra of the products of the glycine, L-histidine, and L-alanine reactions with alpha 2 beta 2 contain contributions from the external aldimine, the quinonoid species, and other intermediates along the catalytic pathway. Just as previously reported for the reaction of L-serine with beta 2 [Goldberg, M. E., York, S., & Stryer, L. (1968) Biochemistry 7, 3662-3667], the reactions of glycine, L-histidine, and L-alanine with the beta 2 form of tryptophan synthase yield spectra with no contributions from catalytic intermediates beyond the external aldimine. The kinetics of intermediate formation and comparisons of the time courses for the exchange of alpha-1H for solvent 2H catalyzed by alpha 2 beta 2 or beta 2 were found to be consistent with these assignments. Intermediates further along the tryptophan synthase catalytic pathway are stabilized to a greater degree in the alpha 2 beta 2 complex than in the beta 2 species alone. This observation strongly suggests that the association of alpha and beta subunits to form the native alpha 2 beta 2 species lowers the activation energies for the interconversion of the external aldimine with chemical species further along the catalytic path.(ABSTRACT TRUNCATED AT 250 WORDS)

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